Programmed cell death is integral to the normal development and homeostasis of many tissues. Genes responsible for programmed cell death have been best defined in the nematode C. elegans, from which two genes required for cell death -- ced-3 and ced-4 -- have been cloned. This proposal is to characterize cell death genes in vertebrate species, using chicken interdigital cell death as a model. Four approaches will be taken to isolate genes that direct the normal physiological program of vertebrate cell death. First, based upon the significant degree to which many genes are evolutionarily conserved, vertebrate homologs of ced-3 and ced-4 will be sought. In preliminary experiments, ced-3 and ced-4 have been cloned from other nematode species, defining regions of these proteins that are conserved. Degenerate oligonucleotides based upon these conserved sequences will be used in PCR experiments to seek similar vertebrate genes. Second, monoclonal antibodies will be isolated that specifically recognize dying cells in chicken interdigital tissue. These antibodies will be used to characterize the process of programmed cell death and will be studied for possible usefulness in analyzing the cell death that occurs in aging and disease. Genes that encode proteins recognized by some of these antibodies will be cloned. One antibody that recognizes dying chicken interdigital cells has already been obtained. Third, genes that are activated during chicken interdigital cell death will be isolated, using the techniques of subtractive and differential screening of cDNA libraries and differential PCR. An activin receptor homolog and a zinc-finger protein have been identified in preliminary screens. Fourth, cell death genes identified in these various ways will be characterized molecularly and biochemically, and their patterns of expression will be determined. These genes will be expressed in cells in culture to see if they induce programmed cell death.